SV40 large T antigen is a multifunctional protein involved in the regulation of viral and cellular gene expression. Mutants have played an important role in studies of the molecular biology of SV40 large T antigen. Previously, a large set of mutants with deletions at Dde I sites in the early region was prepared in this laboratory. Subsequent studies will focus on the transformation potentials of these mutants and the biological and biochemical properties of the mutant large T antigens. One principle aim is to determine whether mutants alone unable to transform primary cells can complement one another, or cloned oncogenes, to transform primary cells. Specific cellular genes are activated by SV40 transformation--additional studies will be directed at determining whether the same cellular genes are also activated in mutant-transformed cell lines. Studies on the oligomerization behavior of mutant large T antigens, their subcellular localization, and their affect on transcription for the SV40 late promoter will also be conducted. Previously, studies in this laboratory determined that the carboxy-terminal portion of large T provides a function needed for efficient growth of SV40 in primary cells or CV-1 (and related) lines. Mutants unable to express this domain are blocked at a late stage of infection, but grow normally in BSC-1 and Vero cells. This domain can be transferred to VP1 and remains functional, indicating that T antigen contains at least 2 separable functional domains. In the coming period, this late function of large T will be studied in detail. Answers to the following questions will be sought: Is the function involved in determining either the tissue or species growth range of SV40? Will mutants unable to express this domain grow in hybrids between permissive (BSC-1) and non-permissive (CV-1) cells? At the molecular level, what defects are exhibited by these mutants? Can mutations at sites distant from the original deletion bring about reversion of the phenotype of these mutants? Major portions of the SV40 A gene, encoding large T antigen, have not yet been studied with sophisticated genetic tools. Therefore, additional mutants will be prepared with small insertions (using oligonucleotide linkers) or deletions at Aha III sites in the SV40 early region. These sites are clustered in portions of the A gene of interest. These mutants will be characterized for their biological and biochemical properties, including their potential for malignant transformation.